Transcriptome Analysis Of Heat Stress Response Genes And Study On Identification And Function Of Heat Shock Transcription Factors In Potato

Potato(Solanum tuberosum L.)is a kind of annual nightshade crop.During potato cultivation in the field,various abiotic stresses would exert detrimental impact on its growth status.Among these abiotic factors,temperature is one of the most important and indisciplinable factors affecting potato growth and development.For potato,the optimal temperature for the growth of aerial portion is 20-25? and the best temperature for tuber formation is 15-20?.Heat stress has a severe impact on potato growth and tuberization process,always resulting in the decrease of tuber yield and quality.Therefore,it is of great significance for potato breeding to illuminate the mechanism of heat stress on potato,reveal the response mechanism of potato to heat stress and explore heat resistant genes.Heat shock transcription factors(Hsfs)play important roles in plant response to high temperature.They transcriptionally regulate the expression of genes encoding heat shock proteins(Hsps)by binding to heat shock element(HSE)in the promoter region of Hsp,The produced Hsps subsequently involve in regulation of heat resistance.Compared with the animal Hsfs,plant Hsfs are more diverse,partially redundant,and functionally flexible.In addition to heat stress,they can respond to other abiotic stresses.At present,the research on Hsfs has been reported in several plant species,including Arabidopsis,tomato,soybean and wheat.However,it has rarely been reported in potato.In this study,the potato leaves under heat stress(35? day/28? night)and under normal condition(20? day/18? night)were used as materials for transcriptome sequencing.According to the RNAseq data,the heat stress response genes were identified in potato.On this basis,the structural characteristics,expression patterns and functional features of potato Hsfs were systematically analyzed.The major results are described as followings:1.Transcriptome analysis of heat stress response genes in potatoTotal RNA was extracted from normal potato leaves(CK)and potato leaves with 3 days of heat treatment(HS),and two cDNA libraries were constructed respectively.Then transcriptome sequencing was performed using Illumina Hiseq-2000 platform.Finally,1420 genes showed significantly differential expression levels between HS and CK cDNA samples.Among these differentially expressed genes(DEGs),the expression of 771 genes was found to be up-regulated and 649 genes down-regulated respectively.The expression patterns of 12 randomly selected genes were detected using droplet digital PCR(ddPCR)to verify the reliability of RNAseq results.The detection results under two different methods had a high correlation,suggesting that the RNA-seq results were reliable for further research.GO enrichment analysis of the differentially expressed genes showed that these DEGs were clustered into 49 different GO types:27 GO terms were assigned to biological process(BP),16 GO terms were assigned to molecular function(MF),and 6 were assigned to cellular component(CC).In the category of biological process,"response to stimulus" was the predominant GO term,which contained 127 DEGs.In the category of molecular function,"catalytic activity" including 310 DEGs was the leading GO term,which contained the most DEGs among these 49 GO terms.In cell component category,"plastid"containing 55 DEGs was the main GO type.These results showed the functional diversity of the heat stress response genes.In order to further investigate the biological pathways in which the differentially expressed genes were involved,these DEGs were mapped to the KEGG database.Totally,110 KEGG pathways were identified.According to the enrichment significance,the most significant 19 pathways were screened out,including "limonene and pinene degradation"(ko00903),"starch and sucrose metabolism"(ko00500),"plant pathogen interaction"(ko04626)and "plant hormone signal transduction"(ko04075).The results indicated that these biological pathways may be involved in heat resistance regulation or be influenced by heat stress.The expression levels of Hsfs and Hsps were analyzed based on the gene expression profile of these DEGs.The results showed that apart from HsfA2 and HsfA3,whose expression levels were significantly decreased,the expression of most Hsfs in potato leaves had no significant difference after 3 days of heat treatment.Although the expression levels of most potato Hsps had no significant difference or were decreased significantly,there still exist some Hsps whose expression levels were markedly increased after 3 days of heat treatment,such as Hsp26-CP and Hsp70.For example,the expression of Hsp26-CP in heat stressed potato leaves was 433-fold higher than that in normal leaves.2.Genome-wide identification and function analyses of heat shock transcription factors in potatoThe previous RNA-seq results revealed that the Hsfs were not efficiently expressed in potato under long-term heat stress(3 days).However,our preliminary experiments showed that the expression of some StHsfs could be induced by short-term heat stress(within 24 hours),indicating that these Hsfs may participate in early heat stress response in potato.Potato Hsf members were searched from the Spud DB Potato Genomics Resources,NCBI GenBank database and Plant Transcription Factor Database(PTFD).Twenty-seven full-length Hsf genes were identified after removing the redundant and non-full length sequences.The variation range of protein length of these Hsfs were from 201 amino acids(StHsf026)to 501 residues(StHsf005),and the theoretical pI varying from 4.71(StHsf014)to 9.58(StHsf026),implying their structure difference and function diversity.Based on the numbers of amino acids inserted in HR-A core and HR-B core of StHsfs,StHsfs were classified into A,B and C three major classes.The phylogenetic analysis of potato helped to classify these Hsf members into several subclasses in comparison with the classification scheme of other species.These StHsfs were mapped to 10 of the 12 potato chromosomes,with no StHsf found on chromosome 1 and chromosome 5.The gene structure diagram depicted that these StHsfs had several differences in intron number,length and position.Twenty conserved motifs of the StHsf proteins were predicted by MEME motif detection software,revealing the conservation and diversification of these proteins in structure and function.The expression patterns of 27 Hsfs in different potato tissues were analyzed using the RNAseq data obtained from Spud DB Potato Genomics Resources(PGSC,2011).The result showed that the majority of StHsfs were expressed in multiple potato tissues,indicating that these genes may participate in different potato biological processes.A few StHsfs exhibited tissue specific expression,such as StHsf015(SthsfA8b)and StHsf017(StHsfA8d),which highly expressed in tubers,indicating that they might be involved in tuber formation and development.Quantitative real-time PCR(qRT-PCR)was used to determine the expression profiles of 18 selected StHsf genes in leaves of potato plants subjected to short-term heat,drought and cold stresses(0 h,2 h,6 h and 24 h).The results showed that these genes have different degrees of response to the three stresses:under heat stress,15 StHsfs were up-regulated and 3 StHsfs were down-regulated;in response to drought stress,3 StHsfs were up-regulated and 15 StHsfs were down-regulated;under cold stress,5 StHsfs were up-regulated and 13 were down-regulated.The expression levels of StHsf004,StHsf005 and StHsf009 induced by heat stress were higher than other members;StHsf007,StHsf009 and StHsf014 had higher expression levels than other members in response to cold stress.In order to further study the potential regulatory relationship between StHsfs and its related genes,a co-expression network was constructed using WGCNA according to the RNAseq data(PGSC,2011).Fifteen co-expression modules containing different StHsfs and their co-expressed genes were formed based on the correlation of biological function among genes.Module 1,which contained StHsf005 and other 642 related genes,was the largest module among the 15 modules.StHsp26-CP and StHsp70 genes,which were effectively expressed after 3 days of heat stress,existed in the same module as StHsfA3,suggesting that StHsp26-CP and StHsp 70 were probably the co-expressed genes of StHsfA3,and their expression might be regulated by HsfA3.These results provide the basis for further investigation of the functional mechanism of StHsfs.3.Analysis of heat resistance in transgenic potato plants overexpressing StHsfA3 and preliminary study on the regulatory relationship between StHsfA3 and its related HspsIn the previous study,we observed that StHsfA3 was highly expressed induced by short-term heat stress,indicating that StHsfA3 may play an important role in enhancing plant heat resistance.StHsp26-CP,StHsp70 and StHsfA3 were in the same co-expression module,suggesting that the expression of StHsp26-CP and StHsp70 may be regulated by StHsfA3.In order to investigate the role of StHsfA3 in enhancing plant heat resistance and explore the regulatory relationship between StHsfA3 and the two Hsps,potato HsfA3 cDNA sequence were cloned from Desiree.The full-length cDNA of StHsfA3 was 1506 bp which encodes 501 amino acids with an estimated molecular weight of 55.23 kDa and a theoretical isoelectric point of 4.9.The vector of StHsfA3-pBAO02 was constructed and transformed into the potato plants via Agrobacteria-mediated genetic transformation.Totally 5 independent transgenic potato plants overexpressing HsfA3 were obtained through the expression detection of StHsfA3 by PCR and qRT-PCR.The relative water content(RWC)and MDA content were assayed to analyze the heat tolerance of transgenic plants.The results showed that the RWC was significantly elevated while the MDA content was significantly decreased in the transgenic plants compared with the non-transgenic plants under heat stress,indicating that StHsfA3 play a positive regulatory role in enhancing the heat resistance of potato.Furthermore,the expression levels of StHsp26-CP and StHsp70 were determined by qRT-PCR in different potato plants.The results exhibited that the expression levels of StHsp26-CP and StHsp70 in the transgenic lines overexpressing StHsfA3 were significantly higher than that in non-transgenic plants,demonstrating that the expression of StHsp26-CP and StHsp70 might be induced by HsfA3.However,interaction relationship between them needs further investigation.